CN101810775B - Chinese medicinal composition for reducing sugar and dripping pills - Google Patents
Chinese medicinal composition for reducing sugar and dripping pills Download PDFInfo
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Abstract
The invention provides a Chinese medicinal composition for reducing sugar, an extract and dripping pills thereof for treating diabetes. The Chinese medicinal composition for reducing the sugar consists of the following components in part by mass: 10 to 20 parts of fiveleaf gynostemma herb, 5 to 15 parts of gardenia, 10 to 30 parts of Chinese yam and 10 to 30 parts of fragrant solomonseal rhizome.The invention has the main advantages that the invention provides the Chinese medicinal composition for reducing the sugar with remarkable sugar reducing effect and good safety and the dripping pillsthereof, which can obviously improve the weight of a diabetes mouse model caused by alloxan, reduce the blood sugar, improve the serum insulin level of a diabetes mouse, reduce the oxidation product MDA content of tissues and improve the content of SOD with remarkable effect; and the Chinese medicinal composition for reducing the sugar and the dripping pills have the advantage of improving the blood sugar, have the capabilities of improving individual oxidation prevention and stress prevention, and can comprehensively treat the diabetes.
Description
(1) technical field
The present invention relates to a kind of blood-sugar lowering tcm drug compositions and drop pill thereof.
(2) background technology
Diabetes (Diabetes mellitus) are a kind of common endocrine metabolism property diseases; Be because insulin deficit or can not normally bring into play physiological action and cause carbohydrate metabolism disturbance in the body; Then causing the disorderly a kind of comprehensive disease of multiple substance metabolism such as protein, fat, water, electrolyte, is principal character with the hyperglycemia.This disease is often followed multiple chronic complicating diseases, causes the extensive injuries of many organs, multisystem, has higher disability rate.According to bibliographical information, global diabetics quantity has surpassed 1.5 hundred million people, and China has glycosuria patient total amount now near 4,000 ten thousand people, and wherein 90% is non-insulin-dependent diabetes mellitus (II type).
The harm of diabetes not only is the just cryptorrheic influence of disease itself; The most serious be the complication that diabetes are brought; Like cardiovascular system diseases, hepatorenal damage and peripheral neuropathy or the like, it has become the continue cardiovascular disease and the later the third-largest cause of disease of cancer.
Main operative treatment and two kinds of methods of hypoglycemic drug of adopting on the treating diabetes aspect doctor trained in Western medicine.Use Chinese medicine and preparation thereof in recent years and prevent and treat the research of diabetes and obtain many new progresses, especially Chinese medicine can overcome chemical synthetic drug in the hypoglycemic fact that has side effects simultaneously or bring out complication, the extremely attention of the world of medicine and extensive patients.Therefore, research Chinese medicine hypoglycemic activity mechanism is developed blood sugar lowering new Chinese medicine quick-acting, efficient, long-acting, low toxicity, has important social value and economic benefit for preventing and treating diabetes and complication thereof.
(3) summary of the invention
The object of the invention provides a kind of blood-sugar lowering tcm drug compositions, extract and drop pill thereof that is used to treat diabetes.
The technical scheme that the present invention adopts is:
A kind of blood-sugar lowering tcm drug compositions, form following component by quality and form (with dry weight basis):
10～20 parts of Herb Gynostemmae Pentaphylli
5～15 parts of Fructus Gardeniaes
10～30 parts of Rhizoma Dioscoreaes
10～30 parts of Rhizoma Polygonati Odorati.
Said composition quality is formed as follows:
15 parts of Herb Gynostemmae Pentaphylli
10 parts of Fructus Gardeniaes
20 parts of Rhizoma Dioscoreaes
20 parts of Rhizoma Polygonati Odorati.
The present composition mainly is made up of Herb Gynostemmae Pentaphylli, Fructus Gardeniae, Rhizoma Dioscoreae, Rhizoma Polygonati Odorati etc.Herb Gynostemmae Pentaphylli supplementing QI and nourishing YIN among the we, the ability of holding concurrently promoting the production of body fluid to quench thirst, heat clearing away, the Fructus Gardeniae clearing away heat and cooling blood is aided with the Rhizoma Polygonati Odorati nourishing YIN and clearing away heat, the effect that the gas of Rhizoma Dioscoreae spleen reinforcing lung is cloudy, four all meet the Chinese medical theory foundation, follow the monarch principle.Chinese medical theory thinks, diabetes belongs to the disease of deficiency of both QI and YIN due to the intense heat due to deficiency of YIN, and deficiency in origin mainly refers to deficiency of kidney-YIN, the real lung dryness of the stomach heat that is meant of mark.So treatment will take into account nourishing YIN and clearing away heat, emphasis is distinguished deficiency and excess, and with the disease typing, in accordance with the law cube, certificate side selects medicine, applies in a flexible way.
Modern study is found: the main pharmacodynamics composition of Herb Gynostemmae Pentaphylli is a gypenoside, it is reported that gypenoside has preventive and therapeutic effect to many-sided diseases such as nerve, blood, circulation, hormonal system and digestive system.Observe the influence of gypenoside to the blood glucose in diabetic rats of streptozotocin moulding, experiment finds that gypenoside can suppress the increase of cholesterol in serum, lipid peroxide, helps blood glucose to reduce.Adopt gypenoside treatment radical damage rat model; Experiment finds that gypenoside can suppress liver and blood plasma lipide peroxidization; Obviously improve the vigor of the erythrocyte superoxide dismutase (SOD) of radical damage rat model and senile rat, have the effect that reduces oxidation and kill radical damage.Observe the influence of gypenoside to mice, rat anti stress ability; Find that gypenoside can make superoxide dismutase, activity of glutathione peroxidase raise; Quicken the removing speed of interior free yl, the protection cellularity exempts from destruction, can improve erythrocyte fortune oxygen function simultaneously; Reduce the accumulation of blood lactic acid, thereby improved the anoxybiotic ability of reply.
Rhizoma Dioscoreae has the effect of spleen reinforcing nourishing the stomach, invigorating the lung and the kidney, can be used for treating diseases such as insufficiency of the spleen chronic diarrhea, chronic enteritis, chronic gastritis, cough and asthma due to lung deficiency, diabetes, seminal emission and enuresis dysentery.Being edible tonic good merchantable brand, is again the medical material of using always.Chinese yam polysaccharide is that Chinese yam polysaccharide is the Rhizoma Dioscoreae main active of generally acknowledging at present.It is reported that Chinese yam polysaccharide has the effect that reduces the alloxan diabetes mouse blood sugar, promote the recovery of diabetic mice body weight.Its mechanism of action maybe with increase insulin secretion, improve the beta Cell of islet function that is damaged and removed polyradical etc. relevant.In addition, Chinese yam polysaccharide also has enhancing human body immunity, defying age, antioxidative effect.
Fructus Gardeniae is bitter, cold in nature, has the effect of pathogenic fire purging relieving restlessness, clearing away heat-damp and promoting diuresis, removing pathogenic heat from blood and toxic substance from the body.The Fructus Gardeniae main chemical compositions has iridoids (Fructus Gardeniae glycoside), flavonoid (gardenin class) etc.From Fructus Gardeniae, extract the effect that the jasminoidin that obtains has blood sugar lowering.In addition, the antiinflammatory action of jasminoidin is strong.The antiinflammatory action that Fructus Gardeniae gives birth to article is the strongest, can obviously suppress the mice auricular concha inflammation due to the Oleum Tiglii, reduction acetic acid induced mice abdominal cavity capillary permeability.
Rhizoma Polygonati Odorati property is sweet, is slightly cold, and attaches to the lung and stomach meridians ability nourishing YIN and moistening the lung, reinforcing stomach reg fluid.Contain polysaccharide, tannin, phlegmatic temperament, cardiac glycoside etc. in the Rhizoma Polygonati Odorati.Domestic also have scholar's research to show that the Rhizoma Polygonati Odorati water extract has tangible reduction to the blood glucose of the inductive diabetic mice of STZ.Warps such as Pan Xingyu are studied the proof Rhizoma Polygonati Odorati extract for many years and are had the inhibition cell-mediated immune responses, suppress the effect of IL-2, the generation of TNF-α inflammation meson.The blood sugar lowering mechanism of Rhizoma Polygonati Odorati extract and blocking-up beta Cell of islet autoimmune response process; It is cell-mediated to suppress the Th1 type; The autoimmune response that comprises secretion IL-2, TNF-α, IFN-γ activate the reaction of local T h2 type, and then it is relevant to secrete Th2 cytokines such as IL-4, IL-10.
A kind of Chinese medicine extract that is used to prepare the medicine of treating diabetes; The raw material of Chinese medicine for preparing said Chinese medicine extract is that mass ratio is 10～20: 5～15: 10～30: 10～30 Herb Gynostemmae Pentaphylli, Fructus Gardeniae, Rhizoma Dioscoreae and Rhizoma Polygonati Odorati; Said Chinese medicine extract is made up of water extract and ethanol extraction, and described water extract and ethanol extraction be preparation as follows respectively:
(1) water extract: take by weighing the Rhizoma Dioscoreae and the Rhizoma Polygonati Odorati of formula ratio, soak 0.5～2h, add the water reflux, extract, 1～3 time of 6～8 times of quality (in Rhizoma Dioscoreae and Rhizoma Polygonati Odorati dry weight sum), each 1～2h; Extracting temperature is 95～100 ℃, removes by filter medicinal residues, merging filtrate; Concentrate, adding ethanol to ethanol volumetric concentration is 80%～90%, behind the placement 24h; Sucking filtration is got the deposition drying and crushing, obtains the water extract powder;
(2) ethanol extraction: take by weighing the Fructus Gardeniae and the Herb Gynostemmae Pentaphylli of formula ratio, soak 0.5～2h, add 65% alcohol reflux 1～2 time of 8～12 times of quality (in Fructus Gardeniae and Herb Gynostemmae Pentaphylli dry weight sum); Each 1～2h removes by filter medicinal residues, merging filtrate; Reclaim ethanol, concentrate; Concentrated solution filters, and gets the deposition drying and crushing, obtains the ethanol extraction powder.
The invention still further relates to the antihypelipidemic drops (to call the compound antihypelipidemic drop pill in the following text) that utilizes said Chinese medicine composition to make, said drop pill is made by following method:
(1) water extract preparation: take by weighing the Rhizoma Dioscoreae and the Rhizoma Polygonati Odorati of formula ratio, soak 0.5～2h, add the water reflux, extract, 1～3 time of 6～8 times of quality, each 1～2h; Extracting temperature is 95～100 ℃, removes by filter medicinal residues, merging filtrate; Concentrate, adding ethanol to ethanol volumetric concentration is 80%～90%, behind the placement 24h; Sucking filtration is got the deposition drying and crushing, obtains the water extract powder;
(2) ethanol extraction preparation: take by weighing the Fructus Gardeniae and the Herb Gynostemmae Pentaphylli of formula ratio, soak 0.5～2h, add 65% alcohol reflux 1～2 time of 8～12 times of quality; Each 1～2h removes by filter medicinal residues, merging filtrate; Reclaim ethanol, concentrate; Concentrated solution filters, and gets the deposition drying and crushing, obtains the ethanol extraction powder;
(3) drop pill preparation: water intaking extract powder and ethanol extraction powder, add the substrate stirring and evenly mixing, pour in the drop pill machine under 70～100 ℃, splash in the condensing agent, cool off, be solidified into ball, drying obtains said antihypelipidemic drops.
The compound antihypelipidemic drop pill has deep clinical accumulation, mainly is made up of Herb Gynostemmae Pentaphylli, Fructus Gardeniae, Rhizoma Dioscoreae, Rhizoma Polygonati Odorati etc.Herb Gynostemmae Pentaphylli supplementing QI and nourishing YIN in the side, the ability of holding concurrently promoting the production of body fluid to quench thirst, heat clearing away, the Fructus Gardeniae clearing away heat and cooling blood is aided with the Rhizoma Polygonati Odorati nourishing YIN and clearing away heat, the effect that the gas of Rhizoma Dioscoreae spleen reinforcing lung is cloudy.More than all medicines share the effect that reaches supplementing QI and nourishing YIN, clearing away heat and promoting production of body fluid.
Condensing agent is the mixture of methyl-silicone oil and liquid paraffin mass ratio 0.5～5: 1 in the said step (3).
Said step (3) mesostroma is PEGE6000, and the quality consumption is water extract powder and ethanol extraction powder gross mass 8～12 times.
Preferably, the raw material of Chinese medicine for preparing said drop pill is that mass ratio is 15: 10: 20: 20 Herb Gynostemmae Pentaphylli, Fructus Gardeniae, Rhizoma Dioscoreae and Rhizoma Polygonati Odorati, and said drop pill is made by following method:
(1) water extract preparation: take by weighing the Rhizoma Dioscoreae and the Rhizoma Polygonati Odorati of formula ratio, soak 1h, add the water reflux, extract, 3 times of 6 times of quality, each 2h; Extracting temperature is 100 ℃, removes by filter medicinal residues, merging filtrate; Being concentrated into relative density is 1.15, and adding ethanol to ethanol volumetric concentration is 90%, behind the placement 24h; Sucking filtration is got the deposition drying and crushing, obtains the water extract powder;
(2) ethanol extraction preparation: take by weighing the Fructus Gardeniae and the Herb Gynostemmae Pentaphylli of formula ratio, soak 1h, add 65% alcohol reflux 2 times of 10 times of quality; Each 2h removes by filter medicinal residues, merging filtrate; Reclaiming ethanol, being concentrated into density is 1.12; Concentrated solution filters, and gets the deposition drying and crushing, obtains the ethanol extraction powder;
(3) drop pill preparation: water intaking extract powder and ethanol extraction powder, adding quality is PEGE6000, the stirring and evenly mixing of 10 times of powder qualities, pours in the drop pill machine under 100 ℃, splashes in the condensing agent, cools off, and is solidified into ball, drying obtains said antihypelipidemic drops; Said condensing agent is methyl-silicone oil and 2: 1 mixture of liquid paraffin mass ratio.
Through the pharmacology test checking, the compound antihypelipidemic drop pill can obviously improve the body weight of the caused diabetic mice model of alloxan, and blood sugar lowering is evident in efficacy, compares with model control group, has utmost point significant difference (P＜0.01); And its drug effect approaches original prescription decoction.The compound antihypelipidemic drop pill can improve diabetic mice serum insulin level; Prompting possibly can protect alloxan relevant to the destruction of mouse islets β cell by the compound antihypelipidemic drop pill; Can stimulate the model mice beta Cell of islet to a certain extent, promote regeneration and the uelralante of islet cells.The compound antihypelipidemic drop pill can reduce the oxidation product MDA content in the tissue, improves the content of SOD, and effect is remarkable, has utmost point significant difference (P＜0.01); Demonstrate fully this prescription and not only have the advantage of improving blood glucose, have more the raising individual oxidation resistance, anti-stress ability, comprehensive treating diabetes.
Chmice acute toxicity preliminary experiment result shows, irritates the stomach animal at the medicinal liquid Cmax, does not cause any visible toxic reaction of mice.Show that preparation can't measure LD50, use its single oral acute toxicity of maximum dosage-feeding test determination instead.Formal experimental result finds, the mouse stomach rear section mice shape that washes one's face, and observing most mice behind the 2h all has laxativeness; Mouse tail point anus ring week is wet; It is thus clear that medicinal liquid shape liquid discharges is arranged, and mice has the shape of tail of licking frequently, analyzing reason possibly be that the mice fasting time is too of a specified duration; Liquor strength and filling stomach amount are too big, cause the GI irritation reaction of short time.Administration 24h observes the mice above-mentioned symptom and disappears, and recovering state is normal.
Viewing duration mice body weight normally increases, physical agility, and activity is freely; The no abnormal symptom of eye; Skin hair color light; The defecation state is normal.See that from the diet curve drug group and blank control group mice diet all have reduction and final trend steadily, this still can not conclude has medicine to cause, need do further long term toxicity test.Experiment finishes respectively to organize mice does not all have death.Under this experiment condition, synthesis result shows that the safety of compound antihypelipidemic drop pill is good.
Beneficial effect of the present invention is mainly reflected in: the invention provides a kind of hypoglycemic effect is remarkable, safety is good blood-sugar lowering tcm drug compositions and drop pill thereof; Can obviously improve body weight, the blood sugar lowering of the caused diabetic mice model of alloxan; Can improve diabetic mice serum insulin level, reduce the oxidation product MDA content in the tissue; Improve the content of SOD, and effect is remarkable; Blood-sugar lowering tcm drug compositions of the present invention and drop pill not only have the advantage of improving blood glucose, have more the raising individual oxidation resistance, anti-stress ability, but comprehensive treating diabetes.
(4) description of drawings
Fig. 1 is that the compound antihypelipidemic drop pill is to tissue of experimental diabetic mice body weight influence (n=8);
Fig. 2 is that the compound antihypelipidemic drop pill is to tissue of experimental diabetic mice blood sugar influence (n=8);
Fig. 3 is that the compound antihypelipidemic drop pill is to tissue of experimental diabetic mice insulin influence (n=8);
Fig. 4 is that the compound antihypelipidemic drop pill is to tissue of experimental diabetic mice SOD influence (n=8);
Fig. 5 is that the compound antihypelipidemic drop pill is to tissue of experimental diabetic mice MDA influence (n=8);
Fig. 6 is that the compound antihypelipidemic drop pill is to tissue of experimental diabetic mice organ index influence (n=8).
(5) specific embodiment
Below in conjunction with specific embodiment the present invention is described further, but protection scope of the present invention is not limited in this:
Embodiment 1: Study on extraction
Instrument, reagent and medical material:
Ultraviolet-uisible spectrophotometer day island proper Tianjin UV-2550
The electric heating constant temperature air dry oven Shanghai one permanent S.C.101 of Science and Technology Ltd. type
Analytical balance Beijing Sai Duolisi balance company limited Sartorius-BS110S
The electronic balance Switzerland METTLER TOLEDO AB135-of company type
The R-204 of Rotary Evaporators Shensheng Science & Tech. Co., Ltd., Shanghai type
The ultrasonic instrument company limited KQ5200 of ultrasonic cleaner city of Kunshan
The Ying Yu of water-bath Gongyi City gives the magnificent HH-S of instrument plant type
High performance liquid chromatograph Waters 510 Series, UV-detector (U.S. Waters Science and Technology Ltd.), chromatographic column: SinoChrom ODS-BP (4.6mm * 250mm, 5 μ m lot numbers: 41021), N2000 chromatographic data work station
Fructus Gardeniae, Herb Gynostemmae Pentaphylli, Rhizoma Polygonati Odorati, Rhizoma Dioscoreae four Chinese medicine material are all available from Chinese Herbal Pieces Factory of Zhejiang Chinese Medical University; Identify through professor Song Jiemin of pharmaceutical college of Zhejiang University of Traditional Chinese Medicine; Fructus Gardeniae is the dry mature fruit of Maguireothamnus speciosus Fructus Gardeniae (Gardenia jasminoides Ellis); Lot number be used as medicine behind the parching to brown: 070717; Herb Gynostemmae Pentaphylli is the herb of cucurbitaceae genus gynostemma Herb Gynostemmae Pentaphylli (Gynostemma P.E.), lot number: 070624; Rhizoma Dioscoreae is the dry rhizome of Dioscoreaceae plant Rhizoma Dioscoreae (Dioscorea opposita Thunb), lot number: 070612; Rhizoma Polygonati Odorati is the dry rhizome of liliaceous plant Rhizoma Polygonati Odorati (Polygonatum odoratum Druce), lot number: 070511
1 Rhizoma Dioscoreae, Rhizoma Polygonati Odorati extraction process are investigated
1.1 investigation index choice
According to documents and materials, polysaccharide component is its important pharmacological component in Rhizoma Dioscoreae and the Rhizoma Polygonati Odorati medical material.In order to give full play to the effectiveness of polysaccharide component in the two flavor Chinese medicines, with polysaccharide component co-extracted in the two flavor medical materials.Generally be to investigate index, so with its evaluation index as polysaccharide extracting process with the polysaccharide yield.
1.2 confirming of quadrature factor level
Take by weighing the medical material of 1 times of recipe quantity (recipe quantity is: Rhizoma Dioscoreae 20g, Rhizoma Polygonati Odorati 20g), totally nine parts, adopt reflux extraction, according to L
9(3
4) Orthogonal Experiment and Design makes an experiment.Extracting liquid filtering concentrates, and carries out the precipitate with ethanol operation by method under the investigation item of extraction solvent.Select solid-liquid ratio, extraction time, extraction time is the investigation factor, and every factor is got three levels, selects L for use
9(3
4) orthogonal table makes an experiment, and sees table 1.
Table 1: factor level table
1.3 orthogonal test checking
Accurately take by weighing the medical material of 1 times of recipe quantity, extract by the orthogonal test optimised process, three parts of parallel extraction, extracting liquid filtering concentrates.Carry out precipitate with ethanol by method under the investigation item of extraction solvent, adopt the phenolsulfuric acid method to measure the content of polysaccharide in the extracting solution, investigate the reasonability of technology.
1.4 the quadrature alcohol precipitation process is investigated
Accurately take by weighing the recipe quantity medical material, press optimum extraction process and extract, extracting solution merges concentrated, according to L
9(3
4) Orthogonal Experiment and Design carries out precipitate with ethanol test.Select cocnentration factor, alcohol precipitation concentration, time of repose are the investigation factor, and every factor is got three levels, selects L for use
9(3
4) orthogonal table makes an experiment, factor level is seen table 2.
Table 2: factor level table
1.5 orthogonal test checking
Accurately take by weighing the medical material of 1 times of recipe quantity, press optimum extraction process and extract, extracting solution merges concentrated, by orthogonal test optimised process precipitate with ethanol, three parts of operation repetitives, according to the content of polysaccharide in the phenolsulfuric acid method mensuration extracting solution, investigates the reasonability of technology.
1.6 result and date processing
Table 3: the orthogonal experiments L that polysaccharide extracts
9(3
4) orthogonal table
Table 4: the orthogonal experiments analysis of variance table that polysaccharide extracts
| Soruces of variation | Degree of freedom | Sum of square of deviations | F | P |
| The A solid-liquid ratio | 2 | 3.872 | 7.375 | ＞0.05 |
| B extraction time | 2 | 21.603 | 41.149 * | ＜0.05 |
| The C extraction time | 2 | 25.135 | 47.876 * | ＜0.05 |
| The D error | 2 | 0.53 | ? | ? |
Annotate: F
005(2,2)=19
Table 5: the orthogonal test confirmatory experiment result (n=3) that polysaccharide extracts
| Sample | Polysaccharide yield (%) | Meansigma methods | RSD(％) |
| 1 | 8.54 | ? | ? |
| 2 | 8.79 | 8.79 | 2.8 |
| 3 | 9.03 | ? | ? |
Table 6: polysaccharide precipitate with ethanol test L
9(3
4) orthogonal table
Table 7: polysaccharide precipitate with ethanol test analysis of variance table
| Soruces of variation | Degree of freedom | Sum of square of deviations | F | P |
| The A cocnentration factor | 2 | 11.455 | 10.587 * | ＜0.10 |
| The B alcohol precipitation concentration | 2 | 10.788 | 9.970 * | ＜0.10 |
| The C time of repose | 2 | 4.152 | 3.837 | ＞0.10 |
| The D error | 2 | 1.08 | ? | ? |
Annotate: F
0.10(2,2)=9
Table 8: polysaccharide precipitate with ethanol verification experimental verification experimental result (n=3)
| Sample | Polysaccharide yield (%) | Average polysaccharide yield (%) | RSD (％) | Yield of extract (%) | Average yield of extract (%) | RSD (％) |
| 1 | 9.28 | ? | ? | 13.11 | ? | ? |
| 2 | 9.57 | 9.40 | 1.6 | 12.93 | 12.87 | 2.1 |
| 3 | 9.36 | ? | ? | 12.57 | ? | ? |
2 Herb Gynostemmae Pentaphylli, Fructus Gardeniae extraction process are investigated
2.1 investigation index choice
According to documents and materials, flavone component is important pharmacological component in saponin constituent in the Herb Gynostemmae Pentaphylli and the jasminoidin medical material.In order to give full play to the effectiveness of active component in the two flavor Chinese medicines,, extract solvent and select ethanol co-extracted in the two flavor medical materials.General Herb Gynostemmae Pentaphylli serves as to investigate index with gypenoside content, and Fructus Gardeniae serves as to investigate index with jasminoidin content, so with gypenoside and the jasminoidin evaluation index as ethanol extraction technology.
2.2 orthogonal extraction technology is investigated
Take by weighing 1 times of recipe quantity medical material (recipe quantity: Herb Gynostemmae Pentaphylli 15g, Fructus Gardeniae 10g), totally nine parts, adopt reflux extraction, make an experiment according to L9 (34) Orthogonal Experiment and Design.Extracting liquid filtering concentrates.With jasminoidin content, gypenoside content is the investigation index, the selective extraction concentration of alcohol, and the solvent amount, extraction time, extraction time is the investigation factor, and every factor is got three levels, selects for use L9 (34) orthogonal table to make an experiment, and factor level is seen table 9.
Table 9: factor level table
2.3 orthogonal test checking
Take by weighing 1 times of recipe quantity medical material, by orthogonal test optimum extraction process, three parts of parallel extraction, extracting liquid filtering concentrates.Measure jasminoidin content and gypenoside content respectively, investigate the reasonability of technology.Accurately draw the concentrated extract 50mL that makes, put water bath method in the constant weight evaporating dish, 105 ℃ of dryings of residue 1 hour move to and cool off 30min in the exsiccator, are operated to constant weight more than the repetition, and accurate the title decides, and calculates yield of extract.
2.4 Herb Gynostemmae Pentaphylli, Fructus Gardeniae extraction process are investigated the result
Table 10:L
9(3
4) orthogonal table
Annotate: (comprehensive grading=jasminoidin content * 70%+ gypenoside content * 30%)
Table 11: analysis of variance table
| Soruces of variation | Degree of freedom | Sum of square of deviations | F | P |
| The A concentration of alcohol | 2 | 145.583 | 7.138 | ＞0.10 |
| B solvent amount | 2 | 20.396 | 1.000 | ＞0.10 |
| The C extraction time | 2 | 240.075 | 11.771 * | ＜0.10 |
| D extraction time | 2 | 40.327 | 1.977 | ＞0.10 |
| Error | 2 | 20.40 | ? | ? |
Annotate: F
0.10(2,2)=9
Table 12: demonstration test result (n=3)
| Sample | Jasminoidin content (mg/ml) | Meansigma methods | RSD (％) | Herb Gynostemmae Pentaphylli total glycosides content (mg/mL) | Meansigma methods | RSD(％) |
| 1 | 36.41 | ? | ? | 92.83 | ? | ? |
| 2 | 34.60 | 35.67 | 2.6 | 93.68 | 95.32 | 3.6 |
| 3 | 36.00 | ? | ? | 99.45 | ? | ? |
Study on extraction is that medical material soaks 1h through single factor and the definite best extraction process by water of Rhizoma Dioscoreae, Rhizoma Polygonati Odorati of orthogonal test, adds 6 times of water gaging reflux, extract, 3 times, extracts 2h at every turn, and extracting temperature is 100 ℃; Best alcohol precipitation process is that extracting solution is concentrated into every milliliter to contain the crude drug amount be 1g, adds 95% ethanol to alcohol precipitation concentration and reaches 90%, leaves standstill 24h, filters, and drying promptly gets.Fructus Gardeniae, the best alcohol extraction process of Herb Gynostemmae Pentaphylli are that medical material soaks 1h, add 10 times of amount 65% ethanol extractions 2 times, extract 2h at every turn, filter, and merge extractive liquid, concentrates, and drying under reduced pressure promptly gets.
Embodiment 2: the preparations shaping technical study
The preferred drop pill moulding process of 1 orthogonal experiment
According to above test, the ratio of ratio, PEG4000 and the PEG6000 of selected medicine and substrate, three factors of material temperature, every factor is selected three levels, adopts L
9(3
4) orthogonal test is carried out preferably the drop pill moulding process, is that screening index is carried out comprehensive grading with outward appearance I (hardness, profile and hangover situation), the different coefficient II of the ball method of double differences, dissolve scattered time limit III, the comprehensive grading value is more little good more.Factor level is seen table 4.
Table 13: factor level table
Pass judgment on formula [55]: R=I/Imax*100*0.4+II/IImax*100*0.3+III/IIImax*100*0.3
Outward appearance: hardness: by hard to soft as to be divided into 1～3 fen; Roundness: must be divided into 1～3 fen to rounding not by rounding; Hangover situation: by well to poor as to be divided into 1～3 fen.Dissolve scattered time limit: according to the inspection of an appendix of Chinese Pharmacopoeia version in 2005 control method disintegration, record dissolves the time of loosing, and promptly gets.The different coefficient of the ball method of double differences: according to weight differential control method inspection under an appendix drop pill of Chinese Pharmacopoeia version in 2005 item, appoint and get drop pill 20 balls, the accurate respectively weight of claiming fixed every ball is calculated the different coefficient of the ball method of double differences (RSD), promptly gets.
The preferred drop pill moulding process of 2 orthogonal experiments
By the orthogonal table experiment arrangement, take by weighing substrate, heating and melting adds the Chinese medicine extract powder, and stirring, mixing are in following system of ice-water bath condition.Result of the test is seen table 14.
Table 14:L
9(3
4) orthogonal table
Result of the test shows that best moulding process is C
1B
3A
1, promptly medicine and substrate ratio are 1: 8, and liquid paraffin and methyl-silicone oil ratio are 1: 2, and temperature of charge is 100 ℃.
3 demonstration tests
By above-mentioned optimum process condition, 3 lot sample article are dripped the system demonstration test, and by following concrete grammar inspection.Presentation quality: size is even, and color and luster is consistent, and rounding has hardness, does not have hangover.Dissolve scattered time limit: press the method inspection under Chinese Pharmacopoeia appendix drop pill item in 2005.Weight differential: press the method inspection under Chinese Pharmacopoeia appendix drop pill item in 2005.
Actual in conjunction with producing, confirm that the compound antihypelipidemic dropping pill technique is a medicine: substrate=1: 10, coolant are liquid paraffin: methyl-silicone oil=1: 2, dripping the system temperature is 100 ℃.
Embodiment 3: the antihypelipidemic drops preparation
Prescription: Herb Gynostemmae Pentaphylli 15g, Fructus Gardeniae 10g, Rhizoma Dioscoreae 20g, Rhizoma Polygonati Odorati 20g
Method:
(1) Chinese medicine extract preparation: get Rhizoma Dioscoreae, the Rhizoma Polygonati Odorati of recipe quantity, behind the immersion 1h, add 6 times of amounts (240g) water reflux, extract, 3 times, extract 2h at every turn; Extracting solution is concentrated into every milliliter, and to contain the crude drug amount be 1g, adds 95% ethanol to ethanol volumetric concentration and reach 90%, leaves standstill 24h, filters, and drying promptly gets the water extract dried powder; Get Fructus Gardeniae, the Herb Gynostemmae Pentaphylli of recipe quantity, behind the immersion 1h, add 10 times of amounts (250g), 65% ethanol extraction 2 times, extract 2h at every turn, filter, merge extractive liquid, concentrates, and drying under reduced pressure promptly gets the ethanol extraction dried powder; Water extract dried powder and ethanol extraction dried powder are mixed, be said Chinese medicine extract;
(2) take by weighing substrate PEGE6000, heating and melting adds step (1) Chinese medicine extract powder, and stirring, mixing are in following system of ice-water bath condition.The Chinese medicine extract powder: substrate=1: 10, coolant are liquid paraffin: methyl-silicone oil=1: 2, dripping the system temperature of charge is 100 ℃.Drop pill ratio of briquetting average out to 98.83%.
Embodiment 4: the compound antihypelipidemic drop pill is to ICR mouse blood sugar, serum insulin, SOD, the exponential influence of MDA, protein and liver
Experimental apparatus, medicine and reagent:
Ultraviolet-uisible spectrophotometer day island proper Tianjin UV-2550 of company
The JA-2003 of balance equipment factory of electronic balance Shanghai Precision Scientific Apparatus Co., Ltd type
The Ying Yu of water-bath Gongyi City gives the magnificent HH-S of instrument plant type
The TDL80-2B of centrifuge Anting Scientific Instrument Factory, Shanghai type
The Qiang Bu of blood sugar detection appearance U.S. Lifescan company is type extraordinarily
The MK-3 of ELIASA Finland Lei Bo group
The vortex mixer Beijing XW-80A of medical apparatus factory type
Alloxan (available from Sigma company)
Insulin test kit (brand USCNLIFE)
Superoxide dismutase test kit (bio-engineering research institute is built up in Nanjing)
Malonaldehyde (MDA) is measured test kit (bio-engineering research institute is built up in Nanjing)
JINQI JIANGTANG PIAN, produce lot number by Longshunrong Pharmaceutical Factory, Tianjin Zhongxin Pharmaceutical Group Co., Ltd: 0703066, specification: every heavy 0.42g, for oral administration.Chinese patent medicine is formed: the Radix Astragali, Flos Lonicerae etc.
Compound antihypelipidemic drop pill extract (making by embodiment 3 steps (1) method) is made into desired concn with distilled water during experiment.Former prescription drug group, by Rhizoma Dioscoreae, Rhizoma Polygonati Odorati, Herb Gynostemmae Pentaphylli, Fructus Gardeniae according to embodiment 2 prescriptions with 6 times of water yield reflux, extract, 3 times, each 2h, extracting solution merge and are concentrated into 0.8g/mL.
70 of protein quantification test kit-Coomassie brilliant blue method (bio-engineering research institute is built up in Nanjing) cleaning level ICR mices, body weight 18～22g, male, provide by Zhejiang University of Traditional Chinese Medicine zoopery center, the normal diet adaptability is fed a week.Animal feed: Mus is used normal diet, available from the Zhejiang Academy of Medical Sciences.The raising condition: temperature is 18～22 ℃, relative humidity 50%～60%.
1 tissue of experimental diabetic mice modelling
Get 70 of male mices, body weight 18～22g, normally raise a week after, 10 of picked at random are as the normal control group.Water 12h is can't help in the fasting after the 8th day of all the other mices, joins 5% alloxan solution by the 100mg/kg tail vein injection at present, causes the experimental diabetes disease model.The tail vein is got blood and is surveyed fasting blood sugar behind 72h, is not less than 11.1mmoL/L as the successful standard of modeling with blood glucose value, deletes the person that do not cause the diabetes model.
2 divide into groups and the administration situation
60 mices choosing the modeling success are divided into 5 groups at random, 12 every group, are model control group, positive controls, former prescription decoction group, compound antihypelipidemic drop pill medicine high and low dose group.
Compound antihypelipidemic drop pill drug group: be divided into high and low two dose groups.By prescription crude drug consumption is 65g/ people/day, presses 65kg/ people and calculates, be i.e. 1.00g crude drug/kg; Be converted into mice crude drug amount by people and animal body surface area ratio, promptly the mice dosage is 6.00g/kg, is low dosage with this 2 multiple dose; Its 8 times is high dose; Use distilled water to be made into concentration respectively before facing use, press the 0.2mL/10g gastric infusion, 1 time/d as 2.4g/mL, 0.6g/mL (in crude drug).
JINQI JIANGTANG PIAN group: specification: the 0.42g/ sheet, clinical dosage: 8.82g (21)/people/day presses 65kg/ people and calculates; Be 0.14g crude drug/kg, be converted into mice crude drug amount by people and animal body surface area ratio, promptly the mice dosage is 0.8g/kg; As the contrast pharmaceutical quantities; Use distilled water to be configured to concentration and be 0.4g/mL, press the 0.2mL/10g gastric infusion, 1 time/d.
Former prescription decoction group: by prescription crude drug consumption is 130g/ people/day; Press 65kg/ people and calculate, promptly 2.00g crude drug/kg is converted into mice crude drug amount by people and animal body surface area ratio; Be mice dosage 12.00g/kg; Face and use distilled water to be made into concentration before using to be 0.6g/mL, to press the 0.2mL/10g gastric infusion, 1 time/d.
Normal control group and model control group: irritate stomach equal-volume distilled water, press the 0.2mL/10g gastric infusion, 1 time/d.See table 15.
Table 15: the compound antihypelipidemic drop pill is to diabetic mice model investigation scheme
| Group | Medicine | Drug level | The administration volume | Dosage |
| ? | ? | (g/mL) | (mL/10g) | (g/10g) |
| The normal control group | Distilled water | - | 0.2 | Equal-volume |
| Model control group | Distilled water | - | 0.2 | Equal-volume |
| Positive controls | Gold stilbene sheet | 0.4 | 0.2 | 0.08 |
| Former side's decoction group | Former prescription soup | 0.6 | 0.2 | 0.12 |
| The antihypelipidemic drops low dose group | Drop pill solution | 0.6 | 0.2 | 0.12 |
| The antihypelipidemic drops high dose group | Drop pill solution | 2.4 | 0.2 | 0.48 |
3 collections of specimens and processing
3.1 collection of specimens
Each is organized mice and all adopts the gastric infusion approach, by the thing that tried that gives corresponding dosage shown in the table 32, and every day 1 time, successive administration 15 days.After the modeling success, every 5d collects mouse blood once, and tail vein blood carries out the mensuration of blood glucose, surveys altogether 3 times.Water is can't help in fasting before the sacrifice of animal, gets blood in eye socket, and is centrifugal, is used to measure insulin; Put to death animal, win liver, weigh, and measure malonaldehyde (MDA), superoxide dismutase (SOD) with liver tissue homogenate.
The preparation of 3.210% liver tissue homogenate
Wash with cold saline 3.2.1 get liver organization, remove washing liquid, filtered juice is wiped away dried, and electronic balance is accurately weighed, and puts into the small beaker of 5～10mL.
3.2.2 get the normal saline of pre-cooling with graduated cylinder, the total amount of normal saline is nine times of piece of tissue, is poured in the small beaker, shreds piece of tissue as early as possible with eye scissors.
3.2.3 the tissue that will shred is introduced in the glass homogenate pipe entirely, fully grinds with manual glass refiner, makes tissue homogenateization.
3.2.4 with homogenate centrifugal 15 minutes with 4000r/min.
3.2.5 get supernatant, be kept at the interior each item index of tissue to be measured in-20 ℃ of refrigerators.
4 observation index
4.1 detection blood glucose
At duration of test, respectively at administration 5 days, after 10 days and 15, adopt U.S.'s Lifescan company blood glucose meter and supporting reagent paper, the tail vein is got the blood measuring blood, and blood mice on same day body weight got in record.
4.2 mensuration serum insulin
Mice is in administration after 15 days, fasting 12 hours, and eye socket is got whole blood 2mL, separation of serum, the centrifugal 10min of rotating speed 4000r/min gets serum and is stored in-20 ℃ of refrigerators, adopts ELISA to survey serum insulin.
4.3 measure SOD, MDA
Get 10% liver tissue homogenate, press the test kit description operation and adopt chemical colorimetry to measure SOD vigor in the hepatic tissue in the 550nm place; Adopt chemical colorimetry according to kit measurement MDA content in the 532nm place; Utilize the Coomassie brilliant blue albuminometry organize in protein measuring.
4.3.1SOD measuring principle
Produce superoxide ion free radical (O through xanthine and xanthine oxidase response system
2-), latter's oxidation azanol forms nitrite, under the effect of developer, presents aubergine, surveys its absorbance with visible spectrophotometer.When containing SOD in the sample, then the superoxide ion free radical there is narrow spectrum inhibitory action, the nitrite of formation is reduced, the absorbance of measuring during colorimetric is lower than the absorbance of control tube, calculates through formula and can obtain the SOD vigor in the sample.
4.3.2 MDA measuring principle
Malonaldehyde in the lipid peroxide catabolite (MDA) can with thiobarbituricacid (TBA) condensation, form red product, at the 532mn place maximum absorption band is arranged.
4.3.3 protein determination principle
Protein molecule has a NH
3 +Group, when henna CBBZ250 developer adds in protein standard liquid or the sample, anion on the CBBZ250 dyestuff and albumen one NH
3 +In conjunction with, make solution become blueness, can calculate protein content through measuring absorbance.
4.4 measure the liver index
After 15 days, cut open the belly and get full liver by fasting 12 hours in administration for mice, weighs, and calculates organ index.
5 experimental results
Table 16: the compound antihypelipidemic drop pill is to tissue of experimental diabetic mice body weight influence (n=8)
Annotate: * and model control group be p＜0.05 relatively; * p＜0.01; Δ and positive controls be p＜0.05 relatively; Δ Δ p＜0.01;
Table 17: the compound antihypelipidemic drop pill is to tissue of experimental diabetic mice blood sugar influence (n=8)
Annotate: * * and model control group be P＜0.01 relatively
Table 18: the compound antihypelipidemic drop pill is to tissue of experimental diabetic mice insulin influence (n=8)
Table 19: the compound antihypelipidemic drop pill is to tissue of experimental diabetic mice SOD influence (n=8)
Annotate: * * and model control group be P＜0.01 relatively
Table 20: the compound antihypelipidemic drop pill is to tissue of experimental diabetic mice MDA influence (n=8)
Annotate: * * and model control group compare P＜0.01 Δ Δ and positive controls compares P＜0.01
Table 21: the compound antihypelipidemic drop pill is to tissue of experimental diabetic mice organ index influence (n=8)
6 brief summaries
In the diabetic animal experimentation, often need set up corresponding animal model.In the preparation animal diabetes model, the most common at present with chemicals or excision pancreas.Chemicals is again with alloxan (ALLoxan; ALX) for commonly used; ALX prepares the method for diabetes model and was used 60 years, is to study human diabetes method preferably at present, and one of mechanism of action that it activates the thing diabetes is that it is in vivo through the kinase whose activity of affecting glucose; Reduce the concentration of glucose transporter and glucokinase messenger RNA, oxidation generates OH, O
2-Free radical makes cell membrane produce lipid peroxidation product, destroy islet cells, thereby the hypoinsulinism of making causes the generation of diabetes.The medication that adopts once or lumbar injection or intravenous injection etc. continuously.The difference of administering mode, ID and animal species is different to the damage of animal subject beta Cell of islet.The present invention gropes through trial test with reference to pertinent literature, and finally confirm administering mode: disposable tail vein injection alloxan, ID are the 100mg/kg body weight.After administration the 3rd～4 day, the infringement of islets of langerhans is bigger.In this experiment, the animal of injection alloxan the 3rd day blood sugar detection behind the injection alloxan is higher than the normal control group, shows that diabetes model successfully sets up.This model stability is good, and success rate is high, stimulates less to mice.
6.1 the compound antihypelipidemic drop pill is to the influence of laboratory animal blood glucose, insulin
Hyperglycemia is the main clinical characteristics of diabetes, and improves carbohydrate metabolism, and blood sugar control to desirable level is one of main purpose of treating diabetes.Result of study shows that the compound antihypelipidemic drop pill can improve the diabetic mice carbohydrate metabolism, has the obvious functions of blood sugar effect.
No matter be type i diabetes or type ii diabetes, secular hyperglycemia level itself also can cause beta Cell of islet infringement, and high glucose level to cause beta Cell of islet toxicity be irreversible.Make chemicals ALX the main damage islet cells, particularly beta Cell of islet of diabetes model, can cause that β cell INS secretory function descends, INS secretes minimizing.And measure serum I NS is the common counter of checking the beta Cell of islet function.Result of study shows that compound antihypelipidemic drop pill high dose group can alleviate the infringement of ALX to mouse islets β emiocytosis function.
6.2 the compound antihypelipidemic drop pill is to the influence of laboratory animal serum lipid overoxidation
Diabetics removes outside the Pass the beta Cell of islet damage has with peroxidating, and insulin resistant is all relevant with oxidative stress with aberrant angiogenesis.Several kinds of antioxidant enzymes that exist in the body can prevent oxidative stress.SOD is one of important antioxidant enzyme, and its major function is to remove superoxide radical.In diabetes, the concentration of plasma SOD and insulin resistant close ties, SOD can particularly express and ability catalysis superoxide anion O at the arterial wall camber at blood vessel
2-Thereby become the infringement that hydrogen peroxide and oxygen alleviate lipid peroxidation, so the SOD level is to weigh the key factor of antioxidant ability of organism size in blood and the tissue.
MDA is one of product of MDA reaction, and MDA content can react the degree of lipid peroxidation in the serum.Lipid is biomembranous main component, when free radical and active oxygen attack biomembrane, can be prone to cause the peroxidating damageization to biomembrane, produces lipid peroxidation product simultaneously.Biomembranous peroxide injury can cause variations such as its permeability increases, mobile decline, memebrane protein dysfunction, influence and disrupting biofilm normal physiological function, thus pair cell causes damage.Result of study shows that the compound antihypelipidemic drop pill has remarkable effect to alleviating diabetic mice body peroxide injury, and this possibly also be the compound antihypelipidemic drop pill to one of important mechanisms of protection beta Cell of islet 26S Proteasome Structure and Function.
Claims (6)
1. blood-sugar lowering tcm drug compositions, form following component by quality and form:
2. Chinese medicine extract that is used to prepare the medicine of treating diabetes; The raw material of Chinese medicine for preparing said Chinese medicine extract is that mass ratio is 15: 10: 20: 20 Herb Gynostemmae Pentaphylli, Fructus Gardeniae, Rhizoma Dioscoreae and Rhizoma Polygonati Odorati; Said Chinese medicine extract is made up of water extract and ethanol extraction, and described water extract and ethanol extraction be preparation as follows respectively:
(1) water extract: take by weighing the Rhizoma Dioscoreae and the Rhizoma Polygonati Odorati of formula ratio, soak 0.5～2h, add the water reflux, extract, 1～3 time of 6～8 times of quality, each 1～2h; Extracting temperature is 95～100 ℃, removes by filter medicinal residues, merging filtrate; Concentrate, adding ethanol to ethanol volumetric concentration is 80%～90%, behind the placement 24h; Sucking filtration is got the deposition drying and crushing, obtains the water extract powder;
(2) ethanol extraction: take by weighing the Fructus Gardeniae and the Herb Gynostemmae Pentaphylli of formula ratio, soak 0.5～2h, add 65% alcohol reflux 1～2 time of 8～12 times of quality; Each 1～2h removes by filter medicinal residues, merging filtrate; Reclaim ethanol, concentrate; Concentrated solution filters, and gets the deposition drying and crushing, obtains the ethanol extraction powder.
3. the antihypelipidemic drops that utilizes the said Chinese medicine composition of claim 1 to make, said drop pill is made by following method:
(1) water extract preparation: take by weighing the Rhizoma Dioscoreae and the Rhizoma Polygonati Odorati of formula ratio, soak 0.5～2h, add the water reflux, extract, 1～3 time of 6～8 times of quality, each 1～2h; Extracting temperature is 95～100 ℃, removes by filter medicinal residues, merging filtrate; Concentrate, adding ethanol to ethanol volumetric concentration is 80%～90%, behind the placement 24h; Sucking filtration is got the deposition drying and crushing, obtains the water extract powder;
(2) ethanol extraction preparation: take by weighing the Fructus Gardeniae and the Herb Gynostemmae Pentaphylli of formula ratio, soak 0.5～2h, add 65% alcohol reflux 1～2 time of 8～12 times of quality; Each 1～2h removes by filter medicinal residues, merging filtrate; Reclaim ethanol, concentrate; Concentrated solution filters, and gets the deposition drying and crushing, obtains the ethanol extraction powder;
(3) drop pill preparation: water intaking extract powder and ethanol extraction powder, add the substrate stirring and evenly mixing, pour in the drop pill machine under 70～100 ℃, splash in the condensing agent, cool off, be solidified into ball, drying obtains said antihypelipidemic drops.
4. drop pill as claimed in claim 3 is characterized in that condensing agent is the mixture of methyl-silicone oil and liquid paraffin mass ratio 0.5～5: 1 in the said step (3).
5. drop pill as claimed in claim 3 is characterized in that said step (3) mesostroma is PEGE6000, and the quality consumption is water extract powder and ethanol extraction powder gross mass 8～12 times.
6. drop pill as claimed in claim 3 is characterized in that: the raw material of Chinese medicine for preparing said drop pill is that mass ratio is 15: 10: 20: 20 Herb Gynostemmae Pentaphylli, Fructus Gardeniae, Rhizoma Dioscoreae and Rhizoma Polygonati Odorati, and said drop pill is made by following method:
(1) water extract preparation: take by weighing the Rhizoma Dioscoreae and the Rhizoma Polygonati Odorati of formula ratio, soak 1h, add the water reflux, extract, 3 times of 6 times of quality, each 2h; Extracting temperature is 100 ℃, removes by filter medicinal residues, merging filtrate; Being concentrated into relative density is 1.15, and adding ethanol to ethanol volumetric concentration is 90%, behind the placement 24h; Sucking filtration is got the deposition drying and crushing, obtains the water extract powder;
(2) ethanol extraction preparation: take by weighing the Fructus Gardeniae and the Herb Gynostemmae Pentaphylli of formula ratio, soak 1h, add 65% alcohol reflux 2 times of 10 times of quality; Each 2h removes by filter medicinal residues, merging filtrate; Reclaiming ethanol, being concentrated into density is 1.12; Concentrated solution filters, and gets the deposition drying and crushing, obtains the ethanol extraction powder;
(3) drop pill preparation: water intaking extract powder and ethanol extraction powder, adding quality is PEGE6000, the stirring and evenly mixing of 10 times of powder qualities, pours in the drop pill machine under 100 ℃, splashes in the condensing agent, cools off, and is solidified into ball, drying obtains said antihypelipidemic drops; Said condensing agent is methyl-silicone oil and 2: 1 mixture of liquid paraffin mass ratio.
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